Strain: Human Gender: female Age: 42-year-old Tissue: peripheral blood mononuclear cells Stage: MCNS in relapse
Extracted molecule
total RNA
Extraction protocol
Total RNA was prepared from PBMC from three patients with MCNS during nephrosis and remission phases, using a TRIzol reagent (Gibco-BRL, Gaithersburg, MD, USA).
Label
Cy3
Label protocol
Fluorescence-labeled antisense RNA was then synthesized by direct incorporation of Cy3-UTP or Cy5-UTP (GE Healthcare Bio-Science Corp., Piscataway, NJ, USA) using 2 mg of total RNA and an RNA Transcript SureLABEL Core Kit (TaKaRa BIO Inc., Tokyo, Japan).
Strain: Human Gender: female Age: 42-year-old Tissue: peripheral blood mononuclear cells Stage: MCNS in remission
Extracted molecule
total RNA
Extraction protocol
Total RNA was prepared from PBMC from three patients with MCNS during nephrosis and remission phases, using a TRIzol reagent (Gibco-BRL, Gaithersburg, MD, USA).
Label
Cy5
Label protocol
Fluorescence-labeled antisense RNA was then synthesized by direct incorporation of Cy3-UTP or Cy5-UTP (GE Healthcare Bio-Science Corp., Piscataway, NJ, USA) using 2 mg of total RNA and an RNA Transcript SureLABEL Core Kit (TaKaRa BIO Inc., Tokyo, Japan).
Hybridization protocol
We prehybridized microarrays for 1h at 42 ºC in a solution containing 5×saline-sodium citrate (SSC) (1×SSC: 0.15 M NaCl, 0.015 M sodium citrate), 0.1% sodium dodecyl sulfate (SDS), and 0.1 mg/ml bovine serum albumin. We washed microarrays at room temperature in distilled water 3 times for 2 min and in 2-propanol for 1 min. We performed hybridization for 16-20 h at 42 ºC in solution containing 5×SSC/0.1% SDS and 10% formamide, and pre-heat-denatured samples (4 mg of each fluorescence-labeled antisense RNA, 10 mg of Human Cot I DNA (Invitrogen), 10 mg of poly dA (GE Healthcare Bio-Science Corp.), and 10 mg of yeast tRNA (Invitrogen)). We then washed microarrays in 2×SSC/0.1% SDS at 42 ºC for 4 min, 0.1×SSC/0.1% SDS at room temperature for 4 min, 0.1×SSC at room temperature for 2 min, 0.1×SSC at room temperature for 1 min, and 0.1×SSC at room temperature for 1 min.
Scan protocol
We obtained fluorescence image of Cy3 and Cy5 dye channels using a GenePix 4000B scanner (http://www.axon.com).
Description
Filgen Array Human25K
Data processing
We used Array-Pro Analyzer Version 4.5 (Media Cybernetics, Inc., Silver Spring, MD, USA) to determine the signal intensity of each spot and its local background on microarrays. We calculated net intensity by subtracting the mean intensity of all pixels within the local background area from the mean intensity of all pixels within the spot areas. We normalized biases in net intensity between the two fluorescent dye channels in a microarray by locally weighted linear regression analysis (lowess normalization) using a software [Yang et al. 2002]. Analyzed data were selected by using MicroArray Data Analysis Tool (Filgen, Inc.). These data were total signal value (Cy3 and Cy5) of 1,000 or more and ratio value of 1.5 or more.
